The broad objectives of this research are to develop novel adenovirus (Ad) vectors for human gene therapy and novel approaches to produce Ad vectors. Recombinant Ad vectors are promising agents for therapeutic gene delivery for both short term and long term treatment of a variety of inherited and acquired diseases. Ad vectors offer a number of important advantages for use in gene therapy including wide virus tropism, the ability to efficiently direct gene expression in dividing and non-dividing cells, the capacity to accommodate large gene inserts, and a readily tractable system to produce virus in abundant quantities. The utility of Ad as a gene therapy vector, however, is hampered by the host immune response including innate, humoral, and cellular responses to virus infection. The use of gutted Ad vectors, that is Ad genomes that lack all viral coding sequences, has signtificantly reduced the host immune response to viral infection and greatly enhanced Ad vector efficacy. Gutted Ad vectors in combination with approaches to modulate the host immune response may overcome the limitations of early generations of Ad vectors for long term gene delivery. Predicated on these ideas are efficient means to generate high titer stocks of gutted Ad vectors. Although selective, current approaches for the generation of gutted Ad vectors are laborious and only moderately efficient. We have developed a novel means to generate gutted Ad vectors utilizing an Ad-AAV (adeno-associated virus) hybrid virus. We also have developed a novel approach to regulate the production of a helper virus required for gutted vector production. Finally, we have established the basis for developing new Ad vectors designed to blunt the host's humoral response to Ad infection. The specific aims of this renewal application are: 1) To develop novel Ad-AAV hybrid viruses for the efficient production of highly infectious gutted Ad vectors and without the requirement for wild type Ad helper virus; 2) To develop novel Ad helper viruses with repressible packaging domains to reduce or eliminate helper virus contamination of gutted Ad vector preparation; 3) To develop a system to pseudo-package Ad5 genomes into capsids of other Ad serotypes to circumvent the host's humoral response to Ad infection.